Introduction: Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a chemogenetic tool widely used to dissect signalling in vitro and in vivo1, of which muscarinic DREADDs - hM1Dq, hM3Dq, hM4Di - are most widely used2. Transmembrane mutations render these receptors largely unresponsive to endogenous acetylcholine, but sensitive to the otherwise inert clozapine-N-oxide (CNO). Recent reports suggest back-metabolised clozapine, rather than CNO, is the muscarinic DREADD activator in vivo1. We therefore investigated clozapine agonism at hM1Dq and hM4Di in vitro, and whether back-metabolised clozapine could be detected in CNO-injected mice.

Conclusions: Clozapine has greater affinity and potency than CNO at muscarinic DREADDs in vitro. Furthermore, CNO was back-metabolism to brain-penetrating clozapine in vivo. Collectively, this suggestsclozapine may be a muscarinic DREADD agonist in vivo.